PROJECT SUMMARY Rapidly dividing cells must produce 2,000 new ribosomes every minute, and ensure that they are fully functional. Escape of incompletely assembled ribosomes into the translating pool underlies the high cancer incidence observed in patients suffering from Diamond Blackfan Anemia, 5q- syndrome and congenital asplenia. One focus of our work has been to dissect quality control mechanisms operational during late stages of assembly of the 40S subunit. In this grant, we will focus on how the last ribosomal protein, Rps26, is incorporated into ribosomes, what its roles during translation are, and the consequences of its absence on protein homeostasis. Furthermore, this analysis will be expanded to two additional ribosomal proteins to describe the full set of contacts in the mRNA channel. In Aim 1, we will dissect one of the final steps in 40S maturation, the release of the assembly factors Nob1 and Pno1, via the activity of the Rio1 kinase. Because Rps26 and Pno1 have a highly overlapping binding site, release of Pno1 is required for incorporation of Rps26. We will use genetic and biochemical experiments to probe the release of Nob1 and Pno1 in a Rio1 and ATP-dependent manner, and test the effect from bypass of the Rio1 kinase on ribosome fidelity. In Aim 2, we will define the mechanisms by which Rps26-deficient ribosomes are produced, and what the role of Rps26 in translation termination and stalling is. These goals will be accomplished through in vivo pulse-chase experiments, analysis of Rps26 levels in ribosomes from different yeast strains, luciferase reporter assays and ribosome footprinting. Finally, in Aim 3, we will use ribosome purifications, RNA-seq analysis, and luciferase reporter assays to analyze the mRNA-specificity of two distinct r-protein-deficient ribosomes, those lacking Rps0 and Rps10. By analyzing these three datasets (including Rps26) together, we will define not just the r-protein-mRNA contacts in the entire mRNA binding channel, but also decipher how haploinsufficiency of different r-proteins leads to both shared as well as highly divergent phenotypes. These experiments take advantage findings and reagents produced during the last funding period, and use a unique combination of genetics, genomics and biochemical experiments to address these fundamental questions.